Counteractive effect of mouse dermal fibroblasts during their adipogenic differentiation against Staphylococcus aureus infection and its mechanisms / 中华皮肤科杂志

Objective:To investigate the counteractive effect of mouse dermal fibroblasts (MdFBs) during their adipogenic differentiation against Staphylococcus aureus infection, and to explore its mechanisms. Methods:MdFBs were obtained from newborn C57BL/6 mice, and their adipogenic differentiation was induced by culture in an adipogenic medium for 48 hours. Real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expression of cathelicidin antimicrobial peptide (CAMP) on days 0-6 during the adipogenic differentiation of MdFBs, and Western blot analysis to determine the protein expression of CAMP in the culture supernatant of MdFBs during their adipogenic differentiation. MdFBs were divided into 4 groups: co-stimulation group stimulated by S. aureus suspensions and cultured in an adipogenic medium, adipogenic control group cultured in an adipogenic medium, S. aureus-stimulation group stimulated by S. aureus suspensions and cultured in a common medium, and control group stimulated by phosphate-buffered saline and cultured in a common medium; Western blot analysis and RT-PCR were conducted to determine the protein and mRNA expression of CAMP. S. aureus (5 × 10 4 CFU/ml) was cultured with the culture supernatant of MdFBs after 5-day adipogenic differentiation (adipogenic group), and the growth activity was evaluated every 2 hours during 10 - 24 hours after the start of co-culture; S. aureus cultured with the culture supernatant of MdFBs in a common medium served as the normal control group, and that cultured with cell-free culture supernatant served as the negative control group. Differences between groups were assessed using unpaired t-test or analysis of variance. Results:Significant differences were observed in the relative mRNA expression of CAMP among different time points (days 0, 1, 2, 4, and 6) during the adipogenic differentiation of MdFBs (1.14 ± 0.74, 68.04 ± 12.72, 683.12 ± 38.06, 1 390.68 ± 226.21, 454.57 ± 204.12, F = 50.08, P < 0.001) ; the CAMP mRNA expression was significantly higher on days 1, 2, 4, and 6 than on day 0 ( t = 9.09, 31.03, 10.63, 3.85, respectively, all P < 0.05), and showed an initial rise and subsequent fall during days 0 - 6. The CAMP protein expression in the culture supernatant of MdFBs peaked on days 2-5 and subsequently decreased. Significant differences were observed in the mRNA and protein expression of CAMP among the control group, S. aureus-stimulation group, adipogenic control group and co-stimulation group (mRNA: 0.08 ± 0.02, 0.38 ± 0.10, 0.49 ± 0.11, 0.80 ± 0.03, respectively, F = 43.25, P < 0.05; protein: 0.433 ± 0.176, 0.574 ± 0.176, 1.007 ± 0.176, 1.217 ± 0.176, respectively, F = 46.79, P < 0.05), and the relative mRNA and protein expression of CAMP was significantly higher in the co-stimulation group than in the adipogenic control group, S. aureus-stimulation group and control group (all P < 0.05). At 10 hours during culture, the growth activity of S. aureus was significantly lower in the adipogenic group (0.053 ± 0.015) than in the normal control group and negative control group (0.109 ± 0.015, 0.106 ± 0.015, t = 11.30, 13.26, respectively, both P < 0.05) ; during 10 - 24 hours, the growth activity of S. aureus also showed a significant decrease in the adipogenic group compared with the normal control group and negative control group (all P < 0.05) . Conclusion:MdFBs secreted CAMP during the adipogenic differentiation, and could inhibit the proliferation of S. aureus.

A preliminary study on Candida albicans-induced pyroptosis of murine bone marrow-derived macrophages / 中华皮肤科杂志

Objective:To investigate the effect of Candida albicans ( C. albicans) on pyroptosis of murine bone marrow-derived macrophages (BMDMs) . Methods:Live-cell imaging was used to observe morphologic changes of in vitro C. albicans-infected BMDMs (multiplicity of infection [MOI] = 50) so as to evaluate whether pyroptosis occurred. Cultured BMDMs were divided into a control group and a C. albicans group, which were treated with phosphate-buffered saline and C. albicans suspensions respectively for 6 hours; then, real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of NOD-like receptor pyrin domain containing 3 (NLRP3), interleukin (IL) -1β and IL-18, and Western blot analysis to determine the protein expression and cleavage levels of NLRP3, caspase-1 and gasdermin D (GSDMD). BMDMs were cultured with C. albicans suspensions for different durations (0, 10, 15, 20, and 25 hours), and enzyme-linked immunosorbent assay was conducted to detect secretion levels of IL-1β and IL-18. Cultured wild-type BMDMs and GSDMD-knockout BMDMs were treated with C. albicans suspensions for 15 minutes, and then rates of phagocytosis of C. albicans by wild-type BMDMs and GSDMD-knockout BMDMs were estimated by flow cytometry; after 6-hour treatment with C. albicans, flow cytometry and lactate dehydrogenase (LDH) release assay were performed to assess mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs. In addition, some wild-type BMDMs and GSDMD-knockout BMDMs were separately divided into blank control group, control group, maximum enzyme activity-sample control group, IL-1β alone group, C. albicans alone group, and IL-1β + C. albicans group, and cell mortality rates were detected by the LDH release assay after treatment with IL-1β and/or C. albicans. Statistical analysis was carried out by using unpaired t test, Kruskal-Wallis test, analysis of variance, and other statistical methods. Results:After in vitro treatment with C. albicans, swelling and ballooning with large bubbles blowing from the plasma membrane occurred in BMDMs, suggesting the occurrence of cell pyroptosis; compared with the control group, the C. albicans group showed significantly increased mRNA expression levels of NLRP3 and IL-1β after 6-hour treatment with C. albicans ( t = 13.02, 17.51, respectively, P = or < 0.001), but no significant change in the IL-18 mRNA expression level ( P = 0.486), and Western blot analysis showed that C. albicans could increase the expression of NLRP3 inflammasomes, as well as cleaved caspase-1 and GSDMD. After the treatment with C. albicans for different durations (0, 10, 15, 20, and 25 hours), the secretion level of IL-1β by BMDMs gradually increased over time ( H = 12.90, P = 0.012), while the secretion level of IL-18 did not significantly change ( F = 0.48, P = 0.753), and the secretion level of IL-1β was significantly lower in the GSDMD-knockout BMDM group than in the wild-type BMDM group ( F = 24.22, P = 0.008). After 15-minute in vitro treatment with C. albicans, the phagocytosis rate of C. albicans was significantly lower in the GSDMD-knockout BMDM group (50.3% ± 1.10%) than in the wild-type BMDM group (58.53% ± 1.19%, t = 5.09, P = 0.007) ; after 6-hour treatment with C. albicans, the cell mortality rate was significantly higher in the GSDMD-knockout BMDM group than in the wild-type BMDM group (flow cytometry: 38.40% ± 0.50% vs. 34.37% ± 0.52%, t = 4.72, P = 0.009; LDH release assay: 22.52% ± 0.18% vs. 12.48% ± 0.15%, t = 42.36, P < 0.001) ; the cell mortality rates of wild-type BMDMs and GSDMD-knockout BMDMs both significantly decreased in the IL-1β + C. albicans groups compared with the C. albicans groups (both P < 0.001) . Conclusion:Pyroptosis could be induced in murine BMDMs after C. albicans infection, which promotes the release of IL-1β and may reduce the mortality rate of macrophages by improving their immune activity.

Effect of different incubation time of aminolevulinic acid on photodynamic inhibition of Propionibacterium acnes biofilms / 中华皮肤科杂志

Objective:To investigate the effect of different incubation time of aminolevulinic acid (ALA) on photodynamic inhibition of Propionibacterium acnes biofilms. Methods:Propionibacterium acnes biofilms were formed in 24-well plates with pre-placed cell slides and 96-well plates. The formation of the biofilm structure was observed by confocal laser scanning microscopy (CLSM) , and the growth activity of the biofilm was assessed by the tetrazolium salt XTT assay. The in vitro successfully constructed biofilm models were divided into 6 groups: negative control group receiving neither ALA treatment nor LED radiation, ALA group incubated with ALA alone for 30 minutes, LED group receiving LED radiation alone, ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group incubated with ALA for 15, 30 and 60 minutes respectively followed by LED radiation. After the treatment, CLSM was performed to observe the biofilm structure, as well as to determine the dead/living bacteria ratio, and XTT assay to assess the growth activity of the biofilm. Differences among groups were analyzed using one-way analysis of variance and least significant difference- t test. Results:CLSM showed that the Propionibacterium acnes biofilm model was successfully constructed in vitro. The dead/living bacteria ratios were 0.90 ± 0.16, 1.75 ± 0.19, and 2.57 ± 0.32 in the ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group respectively, which were significantly higher than the dead/living bacteria ratio in the negative control group (0.31 ± 0.01; t= 55.56, 138.62, 74.64, respectively, all P<0.001) ; the biofilm viability value was significantly lower in the ALA-PDT1 group, ALA-PDT2 group and ALA-PDT3 group (0.35 ± 0.02, 0.26 ± 0.02, 0.18 ± 0.01, respectively) than in the negative control group (0.43 ± 0.00; t= 35.66, 2.64, 110.96, respectively, all P < 0.001) . CLSM showed that the structure of the Propionibacterium acnes biofilm was destroyed under the action of ALA-PDT, and the destruction was aggravated with the prolongation of incubation time of ALA. Conclusion:The prolongation of incubation time of ALA can enhance the inhibitory effect of ALA-PDT on Propionibacterium acnes biofilms.

Mechanisms underlying the resistance of Candida albicans to echinocandin antifungals / 中华皮肤科杂志

With the application of echinocandin antifungals, more and more resistant Candida albicans strains have been detected. It has been reported that mechanisms underlying the resistance of Candida albicans to echinocandin antifungals mainly involve FKS, MSH2 and ERG3 gene mutations, biofilm formation, cellular stress response, compensatory increases in chitin, sphingolipid synthesis, etc. This review focuses on echinocandin resistance-related genes and underlying mechanisms in Candida albicans, which will help to overcome and prevent echinocandin resistance in clinical practice, and explore new therapeutic targets and drugs for Candida albicans infection.

Effect of Aspergillus fumigatus on the autophagic flux in murine macrophages: a preliminary study / 中华皮肤科杂志

Objective:To explore the effect of Aspergillus fumigatus ( A. fumigatus) on the autophagic flux in murine bone marrow-derived macrophages (BMDM) . Methods:Murine BMDM were in vitro cultured with heat-killed A. fumigatus for 0, 0.5, 4, and 12 hours. Then, cellular proteins were extracted, and Western blot analysis was performed to detect the conversion of the key autophagy protein microtubule-associated protein 1 light chain 3 (LC3) -Ⅰ to LC3-Ⅱ, and to determine the protein expression of phosphorylated mammalian target of rapamycin (p-mTOR) Ser2481. Additionally, murine BMDM were in vitro cultured with A. fumigatus alone or in combination with different lysosomal inhibitors, including the cysteine cathepsin inhibitor E-64d + pepstatin, bafilomycin-A1 (BAF-A1) , ammonium chloride (NH 4Cl) , and chloroquine, for 4 or 12 hours. Then, Western blot analysis was performed to investigate the effect of A. fumigatus on newly formed LC3-Ⅱ and basal autophagic flux, and confocal laser scanning fluorescence microscopy to analyze the colocalization of A. fumigatus with LC3 and Rubicon (a RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein) . Experimental results at different treatment time points were analyzed by using unpaired t test, and results of experiments evaluating the effect of two factors ( A. fumigatus spores and autophagosome inhibitors) were analyzed by 2 × 2 factorial analysis. Results:After in vitro co-culture with A. fumigatus for 0.5, 4, 12 hours, Western blot analysis showed that the conversion of LC3-Ⅰ to LC3-Ⅱ increased over time in murine BMDM compared with the control (0 hour) group ( t = 6.58, 3.28, 3.02, respectively, all P < 0.05) , but the protein expression level of p-mTOR (Ser2481) did not significantly differ at different treatment time points ( t = 0.441, 0.477, 0.382, P = 0.682, 0.660, 0.722, respectively) . After 4- and 12-hour in vitro treatment, the accumulation levels of LC3-Ⅱ in BMDM significantly increased in the A. fumigatus + chloroquine group compared with the chloroquine-alone group ( t = 2.13, 2.78, respectively, both P < 0.05) , in the A. fumigatus + NH 4Cl group compared with the NH 4Cl-alone group ( t = 2.92, 2.92, respectively, both P < 0.05) , in the A. fumigatus + BAF-A1 group compared with the BAF-A1-alone group ( t = 2.13, 2.13, respectively, both P < 0.05) , and in the A. fumigatus + E-64d + pepstatin group compared with the E-64d + pepstatin group ( t = 2.13, 2.92, respectively, both P < 0.05) . After 8-hour treatment with calcofluor white-labeled A. fumigatus spores, confocal laser scanning fluorescence microscopy showed that LC3 and Rubicon mainly surrounded A. fumigatus, suggesting their colocalization with A. fumigatus. Conclusion:A. fumigatus can in vitro increase the basal autophagic flux in murine BMDM.

Regulatory effect of Candida albicans hyphae on the key autophagy-related molecule microtubule-associated protein 1 light chain 3 in murine bone marrow-derived macrophages / 中华皮肤科杂志

Objective:To evaluate the effect of Candida albicans ( C. albicans) hyphae on autophagic flux in murine bone marrow-derived macrophages (BMDM) . Methods:BMDM were in vitro stimulated with C. albicans hyphae for 0.5, 4 and 12 hours, and the 0-hour group treated without hyphae served as a control. Western blot analysis was performed to detect the conversion of microtubule-associated protein 1 light chain 3 (LC3) -Ⅰto LC3-Ⅱ, and determine the expression of phosphorylated mechanistic target of rapamycin (p-mTOR) at each time point. Some BMDM were divided into several groups: control group receiving no treatment, hyphae group treated with C. albicans hyphae, lysosomal inhibitor groups treated with different lysosomal inhibitors, including E-64d (a cysteine proteinase inhibitor) + pepstatin (a pepsin inhibitor) , bafilomycin-A1 (BAF-A1) , ammonium chloride and chloroquine, and hyphae combined with lysosomal inhibitor groups treated with lysosomal inhibitors immediately followed by C. albicans hyphae. After 4- or 12-hour treatment, the effect of C. albicans hyphae on basal autophagic flux in murine BMDM was evaluated. Statistical analysis was carried out by using unpaired t test, factorial design analysis of variance and least significant difference- t test. Results:After 0.5-, 4- and 12-hour in vitro treatment with C. albicans hyphae, the conversion of LC3-Ⅰ to LC3-Ⅱ significantly increased in murine BMDM (1.254±0.118, 1.629±0.391, 1.598±0.379, respectively) compared with the 0-hour group (0.983±0.030; t=3.875, 2.856, 2.804, respectively, all P< 0.05) , while there was no significant difference in the protein expression of p-mTOR among the 0-, 0.5-, 4- and 12-hour groups. After 4- and 12-hour in vitro treatment with C. albicans hyphae combined with lysosomal inhibitors E-64d and pepstatin, the accumulation level of LC3-Ⅱ significantly increased in BMDM compared with those treated with E-64d and pepstatin alone ( t=3.691, 6.648, respectively, both P< 0.05) . Compared with the corresponding lysosomal inhibitor groups, the accumulation level of LC3-Ⅱsignificantly increased in BMDM treated with C. albicans hyphae combined with BAF-A1, ammonium chloride or chloroquine for 4 and 12 hours (all P< 0.05) . Conclusion:In vitro treatment with C. albicans hyphae can increase the conversion of LC3-Ⅰto LC3-Ⅱ in the basal autophagic flux in murine BMDM.

Analysis of differences in intestinal microbiome in patients with psoriasis vulgaris by using 16S rRNA gene sequencing / 中华皮肤科杂志

Objective:To investigate differences in intestinal microbiome between adult patients with psoriasis vulgaris and healthy individuals.Methods:Fecal samples were collected from 22 patients with confirmed psoriasis vulgaris and 23 healthy controls in Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College from September 2017 to February 2018. The total DNA of intestinal flora was extracted and amplified, and the next-generation 16S rRNA gene-targeted sequencing was performed to analyze the diversity and distribution of intestinal flora. Species annotation and classification were performed according to Silva database, and rank sum test was used to analyze species differences among samples at different taxonomic ranks; QIIME software (Version 1.9.1) was used to calculate the number of operational taxonomic units (OTUs) and main indices of α diversity (Chao1 index, Shannon index and Simpson index) , and t test to analyze differences in indices; PCoA analysis was performed to analyze the difference in β diversity, and differences in microbial community composition structure were analyzed between the two groups by using permutational multivariate analysis of variance; rank sum test and linear discriminant analysis effect size (LEfSe) analysis were used to evaluate the species difference. Results:No significant difference in the number of OTUs was observed between the psoriasis group (147.55 ± 57.07) and healthy control group (148.96 ± 50.45, t = 0.088, P = 0.930) . In addition, there were no significant differences in the Shannon index, Chao1 index or Simpson index between the psoriasis group (4.08 ± 0.80, 169.52 ± 63.17, 0.87 ± 0.07, respectively) and healthy control group (4.11 ± 0.94, 175.36 ± 53.59, 0.86 ± 0.90, respectively; t = 0.12, 0.34, 0.27, all P > 0.05) . PCoA analysis showed that the first and second principal components explained 49.8% and 15.62%, respectively, of the total variance between the psoriasis group and healthy control group, and permutational multivariate analysis of variance revealed that the β diversity significantly differed between the two groups ( P = 0.011) . Different microbes between the psoriasis group and healthy control group included Firmicutes, Clostridia, Clostridiales, Erysipelotrichales and Erysipelotrichaceae, whose abundance significantly increased in the psoriasis group, as well as Epsilonproteobacteria, Campylobacterales, Campylobacteraceae, Campylobacter, Bacteroidales and Bacteroidaceae, whose abundance significantly increased in the healthy control group. Conclusion:The intestinal microbiome differs between patients with psoriasis vulgaris and healthy individuals, which may serve as potential biomarkers for psoriasis vulgaris.

Effect of 5-aminolevulinic Acid Photodynamics Therapy on Biofilm of / 中国医学科学院学报

To investigate the effect of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) on () biofilm. biofilms were constructed on a cell slide and treated with ALA-PDT.According to different light doses,the biofilms were divided into six groups:ALA-PDT group [ALA-PDT1 (50 J/cm),ALA-PDT2 group (100 J/cm),ALA-PDT3 group (200 J/cm)],ALA-only group (ALA group),light-only group (LED),and a negative control group (ALA-PDT-group).The biofilm structure and the ratio of the dead bacteria/live bacteria were observed using a laser confocal microscope (CLSM).Biofilm viability was measured using the XTT assay. CLSM showed that the biofilm structures of ALA group and LED group were not significantly different from that of ALA-PDT-group,whereas the biofilm structure was more seriously damaged in ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group than in the ALA-PDT-group.The ratios of the dead/live bacteria in ALA-PDT-group,ALA group,LED group,ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group were 0.350±0.033, 0.305±0.046, 0.330±0.032, 1.525±0.439, 2.293±0.148 and 3.092±0.189,respectively.ALA group(=0.003, =1.000)and LED group(=-0.025, =1.000)did not significantly differ from the ALA-PDT-group.However,the ratio of dead/live bacteria in ALA-PDT-group was significantly lower than those in ALA-PDT1 group (=-0.162, <0.001),ALA-PDT2 group (=-0.254, <0.001),and ALA-PDT3 group (=-0.352, <0.001).The values of the XTT assay were were 0.462±0.028,0.465±0.044,0.437±0.047,0.301±0.040,0.207±0.001,and 0.110±0.007,respectively,in ALA-PDT-group,ALA group,LED group,ALA-PDT1 group,ALA-PDT2 group,and ALA-PDT3 group.Although the values of XTT assay in ALA(=-0.044, =1.000)and LED groups (=-0.020, =1.000)did not significantly differ from that in ALA-PDT-group,it was significantly higher in ALA-PDT-group than in ALA-PDT1 group (=1.175, <0.001),ALA-PDT2 group (=1.942, <0.001),and ALA-PDT3 group (=-0.352, =2.742, <0.001). ALA-PDT has an inhibitory effect on biofilm.ALA-PDT destroys biofilm structure and inhibits biofilm viability.

Effect of Aspergillus fumigatus on the expression of tumor necrosis factor-α and activation of intracellular signaling molecule p38 mitogen-activated protein kinase by a human acute monocytic leukemia cell line THP-1 / 中华皮肤科杂志

Objective To evaluate the effect of Aspergillus fumigatus on the expression of tumor necrosis factor-α (TNF-oα) and activation of intracellular signaling molecule p38 mitogen-activated protein kinase (p38MAPK) in a human acute monocytic leukemia cell line THP-1.Methods Cultured THP-1 cells (2 x 105/ml) were divided into 4 groups to be treated with Aspergillus fumigatus suspensions at concentrations of 106 and 107 colony-forming units (CFU)/ml (106-and 107-CFU/ml Aspergillusfumigatus groups),100 mg/L β-glucan (a positive stimulus,β-glucan group),culture medium (blank control group) respectively for 1,3 and 6 hours.Real-time fluorescence-based quantitative PCR (qPCR) was conducted to determine the mRNA expression of TNF-α in the THP-1 cells in the above groups.Some other THP-1 cells were treated with 107 CFU/ml Aspergillusfumigatus suspensions (107-CFU/ml Aspergillusfumigatus group),β-glucan (β-glucan group) and culture medium (blank control group) separately for 24 hours,and enzymelinked immunosorbent assay (ELISA) was performed to detect the level of TNF-α in the culture supernatant of THP-1 cells.Western blot analysis was conducted to detect the levels of p38MAPK and phosphorylated p38MAPK in THP-1 cells after 15-,30-and 60-minute treatment with 107 CFU/ml Aspergillusfumigatus suspensions.After 2-hour incubation with the p38MAPK inhibitor SB203580 (20 μmol/L),some THP-1 cells were additionally treated with 107 CFU/ml Aspergillus fumigatus suspensions,β-glucan and culture medium separately for 6 hours,and those without SB203580 treatment served as the control group.Then,qPCR was performed to measure the mRNA expression of TNF-α in the THP-1 cells in the above groups.Results The mRNA expression of TNF-α significantly differed among the 106-and 107-CFU/ml Aspergillus fumigatus groups,β-glucan group and blank control group (F =110.983,P ＜ 0.001),and significantly increased over time (F =701.680,P ＜ 0.001).After 24-hour treatment with 107 CFU/ml Aspergillus fumigatus suspensions,the TNF-α level(6 236.30 ± 437.12 ng/L)significantly increased compared with the blank control group (132.10 ± 0.61 ng/L,P ＜ 0.01).Thirty minutes after the treatment with 107 CFU/ml Aspergillusfumigatus suspensions,the phosphorylated p38MAPK level significantly increased,but started to decrease at 60 minutes.The mRNA expression of TNF-α was significantly lower in the SB203580-treated Aspergillusfumigatus groups (3.83 ± 0.62) than in the SB203580-untreated Aspergillus fumigatus groups (187.23 ± 21.62).Conclusion After the treatment with Aspergillus fumigatus,human THP-1 cells can activate the signal molecule p38MAPK and secrete TNF-α,suggesting that monocytes may participate in the innate immune response to Aspergillusfumigatus infection.

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1 / 中华皮肤科杂志

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.

Expression of interleukin-6 in cystic lesions of patients with acne vulgaris and in vitro effect of Propionibacterium acnes on the production of interleukin-6 by human THP-1 monocytes / 中华皮肤科杂志

Objective To determine the expression of interleukin-6 (IL-6) in cystic lesions of patients with acne vulgaris,and to evaluate the in vitro effect of Propionibacterium acnes (P.acnes) on the production of IL-6 and activation of p38 mitogen-activated protein kinase (p38MAPK) in the human acute monocytic leukemia cell line THP-1.Methods Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of IL-6 in cystic lesions of 6 patients with acne vulgaris,as well as in skin tissues of 6 healthy persons.Some cultured THP-1 cells were divided into 5 groups to be treated with 2 × 106 CFU/ml,2 × 107 CFU/ml and 2 × 108 CFU/ml heat-killed P.acnes suspensions (P.acnes groups),100 μμtg/L lipopolysaccharide (LPS group) and RPMI 1640 medium (control group) respectively.After 1-,3-and 6-hour treatment,real-time fluorescence-based quantitative PCR was conducted to determine the mRNA expression of IL-6 in the above groups.Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of IL-6 in the culture supernatant of cells in the 2 × 108-CFU/ml P.acnes group,LPS group and control group at 24 hours after the treatment.Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK in the 2 × 108-CFU/ml P.acnes group after 15-,30-and 60-minute treatment,as well as in the LPS group after 30-minute treatment and in the control group.Some other THP-1 cells were divided into 3 groups:2 × 108-CFU/ml P.acnes group treated with 2 × 108 CFU/ml P.acnes suspensions,SB203580 (an inhibitor of p38MAPK) group treated with 20 μmol/L SB203580 for 30 minutes followed by the treatment with 2 × 108 CFU/ml P.acnes suspensions,and control group treated with RPMI 1640 medium alone.After 6-hour treatment,the mRNA expression of IL-6 in the above 3 groups was measured by real-time fluorescencebased quantitative PCR.Results The mRNA expression of IL-6 was significantly higher in the cystic lesions of acne vulgaris than in the normal skin tissues (3.680:±:0.790 vs.1.155 ± 0.250,t =3.047,P ＜0.05).Two-way analysis of variance showed that there were significant difference in the mRNA expression of IL-6 among the 2 × 106-CFU/ml,2 × 107-CFU/ml and 2 × 108-CFU/ml p.acnes groups,LPS group and control group (F =532.3,P ＜ 0.001,v =4),and the mRNA expression of IL-6 significantly differed among different time points (F =526.6,P ＜ 0.001,v =2).There were also significant differences in the IL-6 level in the culture supernatant of cells among the 2 × 108-CFU/ml p.acnes group ([1 618.22 ± 32.23] ng/L),LPS group ([3 212.06 ± 353.00] ng/L) and control group ([147.10 ± 0.53] ng/L;v =2,F =102.35,P ＜0.01).After 15-,30-and 60-minute treatment with 2 × 108 CFU/ml P.acnes suspensions,the protein expression of phosphorylated p38MAPK obviously increased.The mRNA expression of IL-6 in THP-1 cells was significantly lower in the SB203580 group than in the 2 × 108-CFU/ml p.acnes group (t =15.91,P =0.004).Conclusions The mRNA expression of IL-6 evidently increases in the cystic lesions of patients with acne vulgaris.P.acnes can activate the signaling molecule p38MAPK in THP-1 cells,and promote the production of IL-6 by THP-1 cells.

Dynamic distribution of calmodulin labeled with red fluorescent protein in the polar growth of Aspergillus fumigatus / 中华皮肤科杂志

Objective To construct a native promoter-regulated Aspergillus fumigatus strain containing red fluorescent protein-labeled calmodulin (CaM-RFP),and to observe the dynamic distribution of calmodulin during the growth of Aspergillus fumigatus.Methods Bilateral flanking sequences of Aspergillus fumigatus calmodulin gene were designed,and plasmids containing the two flanking sequences and mRFP-Aspergillus fumigatus pyrG gene (mRFP-AfpyrG) were amplified separately.The final linear PCR product for transformation was generated from the above three PCR products by fusion PCR.Then,the above linear fragment was transferred into the Aspergillus fumigatus strain by protoplast transformation,so as to construct the CaM-RFP Aspergillus fumigatus strain.The monoclonal colony was picked from the screening medium and subjected to culture.Then,the stablest fluorescent monoxenic strain of Aspergillus fumigatus was selected,and the transformant was verified by PCR.The recombinant strain and wild-type stain were cultured on solid nutrient media separately,and the morphology of these strains was observed by fluorescence microscopy at different time points.Additionally,the above 2 strains were cultured in liquid media separately,and XTT assay was performed to evaluate the growth activity of strains.Microscopy was also conducted to dynamically observe the CaM-RFP Aspergillusfumigatus strain,and analyze the spatial and temporal distribution of calmodulin during the growth and development of Aspergillus fumigatus.Results The fluorescent phenotype and PCR identification results both indicated the successful construction of the CaM-RFP Aspergillus fumigatus strain.The growth activity at 24 hours did not differ between the recombinant strain and wild-type stain (A490:0.689 ± 0.081 vs.0.678 ± 0.054,t =1.32,P ＞0.05),so did the morphology.During the polarized growth of Aspergillus fumigatus,calmodulin was always at the top of the hyphae,germination site of the hyphal branch and the top of new branches.Conclusion Calmodulin may be involved in the regulation of spore germination and polar hyphal growth of Aspergillus fumigatus.

Effect of amphotericin B on the production of tumor necrosis factor-a and interleukin-8 and activation of signaling molecule p38MAPK in human THP-1 cells / 中华皮肤科杂志

Objective To evaluate the effect of amphotericin B on the production of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) and activation of p38 mitogen-activated protein kinases (p38MAPK) in a human acute monocytic leukemia cell line (THP-1).Methods Cultured THP-1 cells were divided into several groups:blank control group receiving no treatment,amphotericin B groups treated with 2,4 and 8 mg/L amphotericin B separately,positive control group treated with 100 μg/L β-glucosan or 100 mg/L lipopolysaccharide.Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of TNF-α and IL-8 after the THP-1 cells were treated with different stimuli for some durations.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the level of TNF-α in the culture supernatant of THP-1 cells after 24-hour treatment with 8 mg/L amphotericin B,and Western blot analysis to measure the levels of p38MAPK and phosphorylated p38MAPK after 30-minute treatment with 8 mg/L amphotericin B.Results After 6-hour treatment with 2,4 and 8 mg/L amphotericin B separately,the mRNA levels of TNF-α in THP-1 cells (7.55 ± 1.17,19.47 ± 2.91,57.22 ± 0.65) and IL-8 (2.98 ± 0.04,5.22 ± 1.35,11.82 ± 1.66) were all significantly higher than those in the blank control group (TNF-α:1.00 ± 0.07,P ＜ 0.01,0.001,0.001 respectively;IL-8:1.01 ± 0.23,P ＜ 0.01,0.001,0.001 respectively).After the treatment with 8 mg/L amphotericin B for 1,3,6 hours,the mRNA levels of TNF-α (8.61 ± 0.30,10.75 ± 0.08,56.98 ± 2.43) and IL-8 (2.63 ± 0.28,5.35 ± 0.98,11.73 ± 1.18) in THP-1 cells were all significantly higher than those in the blank control group (TNF-α:1.18 ± 0.17,P ＜ 0.05,0.01,0.001;IL-8:1.23 ± 0.11,P ＜ 0.05,0.01,0.001).After 24-hour treatment with 8 mg/L amphotericin B,the level of TNF-α in the culture supernatant of THP-1 cells was significantly higher than that in the blank control group (4 039.06 ± 223.87 ng/L vs.96.31 ± 0.26 ng/L,P ＜ 0.001).Conclusion Amphotericin B can promote the p38MAPK phosphorylation and increase the levels of TNF-α and IL-8 in human THP-1 cells in vitro,suggesting its immunomodulatory effects.

Effect of amphotericin B on the production of tumor necrosis factor-a and interleukin-8 and activation of signaling molecule p38MAPK in human THP-1 cells / 中华皮肤科杂志

Objective To evaluate the effect of amphotericin B on the production of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) and activation of p38 mitogen-activated protein kinases (p38MAPK) in a human acute monocytic leukemia cell line (THP-1).Methods Cultured THP-1 cells were divided into several groups:blank control group receiving no treatment,amphotericin B groups treated with 2,4 and 8 mg/L amphotericin B separately,positive control group treated with 100 μg/L β-glucosan or 100 mg/L lipopolysaccharide.Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of TNF-α and IL-8 after the THP-1 cells were treated with different stimuli for some durations.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the level of TNF-α in the culture supernatant of THP-1 cells after 24-hour treatment with 8 mg/L amphotericin B,and Western blot analysis to measure the levels of p38MAPK and phosphorylated p38MAPK after 30-minute treatment with 8 mg/L amphotericin B.Results After 6-hour treatment with 2,4 and 8 mg/L amphotericin B separately,the mRNA levels of TNF-α in THP-1 cells (7.55 ± 1.17,19.47 ± 2.91,57.22 ± 0.65) and IL-8 (2.98 ± 0.04,5.22 ± 1.35,11.82 ± 1.66) were all significantly higher than those in the blank control group (TNF-α:1.00 ± 0.07,P ＜ 0.01,0.001,0.001 respectively;IL-8:1.01 ± 0.23,P ＜ 0.01,0.001,0.001 respectively).After the treatment with 8 mg/L amphotericin B for 1,3,6 hours,the mRNA levels of TNF-α (8.61 ± 0.30,10.75 ± 0.08,56.98 ± 2.43) and IL-8 (2.63 ± 0.28,5.35 ± 0.98,11.73 ± 1.18) in THP-1 cells were all significantly higher than those in the blank control group (TNF-α:1.18 ± 0.17,P ＜ 0.05,0.01,0.001;IL-8:1.23 ± 0.11,P ＜ 0.05,0.01,0.001).After 24-hour treatment with 8 mg/L amphotericin B,the level of TNF-α in the culture supernatant of THP-1 cells was significantly higher than that in the blank control group (4 039.06 ± 223.87 ng/L vs.96.31 ± 0.26 ng/L,P ＜ 0.001).Conclusion Amphotericin B can promote the p38MAPK phosphorylation and increase the levels of TNF-α and IL-8 in human THP-1 cells in vitro,suggesting its immunomodulatory effects.

Effects of the yeast form of Sporothrix schenckii on activation of p38MAPK and expression of interleukin-6 in human THP-1 macrophage-like cells / 中华皮肤科杂志

Objective To evaluate effects of the yeast form of Sporothrix schenckii on activation of p38 mitogen-activated protein kinase (p38MAPK) and expression of interleukin-6 (IL-6) in macrophagelike THP-1 cells,which were differentiated from the human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii at a concentration of 2 × 106 colony-forming units (CFU)/ml (yeast form group),100 mg/L curdlan (curdlan group) and RPMI 1640 medium (blank control group) respectively.Real-time fluorescence-based quantitative PCR was performed to measure the mRNA expression of IL-6 in THP-1 macrophage-like cells in the above 3 groups after 3-and 6-hour treatment separately,and enzyme-linked immunosorbent assay (ELISA) to detect the level of IL-6 in the culture supernatant of THP-1 macrophagelike cells after 24-hour treatment.Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK (p-p38MAPK) in the above 3 groups after 30-and 60-minute treatment separately.Other THP-1 macrophage-like cells were pretreated with 100 nmol/L dexamcthasonc (a p38MAPK inhibitor) for 30 minutes,and then were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii,curdlan and RPMI 1640 medium respectively,and changes in the level of pp38MAPK and mRNA expression of IL-6 were also detected.Statistical analysis was carried out with SPSS19.0 software by using one-way or multi-way analysis of variance and least significant difference (LSD) test.Results Significant differences in the mRNA expression of IL-6 in THP-1 macrophage-like cells were observed among the yeast form group,curdlan group and blank control group (F =5 552.22,P ＜0.001) after 3-hour treatment (56.81 ± 7.36,26.69 ± 1.22 and 0.97 ± 0.05,respectively) and 6-hour treatment (378.03 ± 16.67,276.24 ± 39.13 and 1.02 ± 0.04,respectively).Additionally,the yeast form group showed significantly higher mRNA expression of IL-6 after 6-hour treatment than that after 3-hour treatment (q =16.74,P ＜ 0.001).After 24-hour treatment,the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells also significantly differed among the yeast form group,curdlan group and blank control group (59.96 ± 18.16 pg/L,91.01 ± 17.27 pg/L,5.50 ± 2.30 pg/L,respectively;F =26.62,P ＜ 0.01),and was significantly higher in the yeast form group than in the blank control group (P ＜ 0.01).After 30-and 60-minute treatment,the protein expression of p-p38MAPK was significantly higher in the yeast form group than in the blank control group (both P ＜ 0.01).Moreover,the mRNA expression of IL-6 (4.46 ± 1.03 vs.493.52 ± 113.87,P ＜ 0.001) and protein expression of p-p38MAPK (2.29 ± 0.37 vs.4.55 ±0.46,q =10.81,P ＜ 0.01) were both significantly lower in the yeast form group with dexamethasone pretreatment than in that without dexamethasone pretreatment.Conclusion In vitro treatment with the yeast form of Sporothrix schenckii can enhance the expression of IL-6 in human THP-1 macrophage-like cells by activating the p38MAPK signaling pathway.

Effects of Candida albicans on the expression of tumor necrosis factor-α and activation of the intracellular signaling molecule p38MAPK in a human acute monocytic leukemia cell line THP-1 / 中华皮肤科杂志

Objective To investigate the effects of Candida albicans on the expression of tumor necrosis factor-α(TNF-α)and activation of the intracellular signaling molecule p38 mitogen-activated protein kinase(p38MAPK)in a human acute monocytic leukemia cell line THP-1. Methods Some THP-1 cells were divided into several groups in vitro: two C. albicans groups treated with 105 CFU/ml and 106 CFU/ml heat-killed C. albicans respectively, a lipopolysaccharide (LPS)group treated with 100 μg/L LPS, a blank control group treated with RPMI 1640 medium, two dexamethasone-inhibited groups pretreated with 40 μg/L dexamethasone for 30 minutes followed by treatment with 106 CFU/ml heat-killed C. albicans and LPS respectively. After treatment for 1, 3 and 6 hours, real-time fluorescence-based quantitative PCR was performed to measure TNF-α mRNA expression in THP-1 cells in the above groups. Enzyme-linked immunosorbent assay(ELISA)was conducted to determine the level of TNF-α protein in the supernatant of THP-1 cells treated with 106 CFU/ml heat-killed C. albicans, 100 μg/L LPS or RPMI 1640 medium(blank control group)for 24 hours. Western blot was performed to measure the protein expression of p38MAPK and phosphorylated p38MAPK in THP-1 cells after treatment with 106 CFU/ml heat-killed C. albicans or RPMI 1640 medium (blank control group)for 30 and 60 minutes. Statistical analysis was carried out by using two-way analysis of variance, one-way analysis of variance and the least significant difference(LSD)-t test. Results Significant differences were observed in the mRNA expression level of TNF-α among the C. albicans groups, LPS group and blank control group (F = 110.98, P < 0.001). The mRNA expression level of TNF-α in THP-1 cells increased over time in a time-dependent manner after C. albicans treatment, with significant differences among different time points (F = 701.680, P < 0.001). Compared with the blank control group, both 106-CFU/ml C. albicans group and LPS group showed a significant increase in TNF-α protein expression (6385.70 ± 533.99 ng/L and 3212.06 ± 353.00 ng/L vs. 147.10 ± 0.53 ng/L, P < 0.001 and 0.005, respectively). An obvious increase was observed in the expression level of phosphorylated p38MAPK protein, but no significant changes were noted in that of p38MAPK protein, in THP-1 cells treated with 106 CFU/ml C. albicans for 30 and 60 minutes compared with the blank control group. The mRNA expression level of TNF-α significantly decreased in dexamethasone-pretreated 106-CFU/ml C. albicans group and LPS group compared with those without dexamethasone pretreatment(3.77 ± 0.62 vs. 208.50 ± 10.50, 6.20 ± 1.93 vs. 161.35 ± 1.65, both P < 0.001). Conclusions Heat-killed C. albicans can induce the activation of p38MAPK in and secretion of TNF-α by human THP-1 cells, which then participate in the innate immune response against C. albicans.

Research Advances in Analysis of Trace Mercury in Water / 环境与健康杂志

The methods of separation, enrichment, and analysis of trace mercury in water samples in recent years were reviewed in the present paper. Many methods can be used to analyze trace mercury after separation and enrichment. Spectrophotometry is cheap and simple. Atomic fluorescence spectrometric method is accurate in quantitative analysis. The chromatography method is usually used in the morphological analysis of mercury. Multistep analysis of mercury can be done by joint methods in one time.